Kv3.4 regulates cell migration and invasion through TGF-β-induced epithelial–mesenchymal transition in A549 cells

Epithelial–mesenchymal transition (EMT) is the process by which epithelial cells acquire mesenchymal characteristics. This process induces cell migration and invasion, which are closely related to cancer metastasis and malignancy. EMT consists of various intermediate states that express both epithelial and mesenchymal traits, called partial EMT. Recently, several studies have focused on the roles of voltage-gated potassium (Kv) channels associated with EMT in cancer cell migration and invasion. In this study, we demonstrate the relationship between Kv3.4 and EMT and confirm the effects of cell migration and invasion. With TGF-β treatment, EMT was induced and Kv3.4 was also increased in A549 cells, human lung carcinoma cells. The knockdown of Kv3.4 blocked the EMT progression reducing cell migration and invasion. However, the Kv3.4 overexpressed cells acquired mesenchymal characteristics and increased cell migration and invasion. The overexpression of Kv3.4 also has a synergistic effect with TGF-β in promoting cell migration. Therefore, we conclude that Kv3.4 regulates cancer migration and invasion through TGF-β-induced EMT and these results provide insights into the understanding of cancer metastasis.


blocks EMT progression with reducing cell migration and invasion
Using small interfering RNA of Kv3.4 (siKv3.4),we investigated the knockdown effect of Kv3.4.After 50 nM of siKv3.4 for 72 h in A549 cells, we examined the changes in EMT markers on mRNA and protein levels (Fig. 3A-C).The transfection of siKv3.4 did not affect cell viability (Supplementary Fig. 1B).In mRNA levels, E-cadherin, an epithelial marker, was significantly increased, but vimentin, a mesenchymal marker, was remarkably decreased.N-cadherin, a mesenchymal marker, was not altered in the mRNA levels (Fig. 3A).On the other hand, according to the western blot data, the expression levels of E-cadherin and N-cadherin were slightly decreased, but vimentin expression did not change.In addition, the expression levels of the transcriptional factors such as Snail, Slug, and Twist were significantly reduced (Fig. 3B,C).From these results, we conclude that the reduction of Kv3.4 decreases the mesenchymal markers coexisting with E-cadherin.To further investigate the functions of siKv3.4,we performed the transwell migration and invasion assay after siKv3.4treatment.Our data showed that siKv3.4treatment significantly inhibited cell migration and invasion (Fig. 3D-G).

The overexpression of Kv3.4 acquires mesenchymal characteristics and enhances cell migration and invasion
To explore the role of Kv3.4 expression, we overexpressed Kv3.4 using the KCNC4 overexpression vector and confirmed it through mRNA and protein analysis in A549 cells (Fig. 4A-C).The overexpression of Kv3.4 apparently exhibited morphological changes.Compared with control, Kv3.4 overexpressed cells had prominently elongated shapes and decreased the circularity index, which is closely related to mesenchymal phenotypes (Fig. 4D,E).Furthermore, we assessed the expression levels of EMT markers in the mRNA and protein levels (Fig. 4F-H).N-cadherin significantly increased both mRNA and protein levels.Vimentin also increased mRNA level but did not alter the protein level.However, the expression of E-cadherin remained continuous in mRNA and protein levels.To examine the functional effects of Kv3.4 overexpression, we performed transwell migration (Fig. 4I,J) and invasion assay (Fig. 4K,L).The numbers of migrated and invaded cells were significantly increased by the overexpression of Kv3.4,compared with control.From these data, we conclude that the overexpression of Kv3.4 acquires mesenchymal characteristics retaining the E-cadherin, which enhances cell migration and invasion.

The treatment of TGF-β in Kv3.4 overexpressed cells has a synergistic effect on cell migration
To examine whether TGF-β treatment had a synergistic effect with Kv3.4 overexpression, we treated 2 ng/ml of TGF-β for 24 h after overexpression of Kv3.4 in A549 cells (Fig. 5A).As mentioned before (Fig. 1B), TGF-β treated cells had elongated spindle shapes compared with the cuboidal shapes of control.Kv3.4 overexpressed cells with TGF-β treatment (Fig. 5B,C, OT) had also apparently more elongated and spindle-like morphology and reduced the circularity index compared with other control groups: empty control group (Fig. 5B,C, EC), only Kv3.4 overexpressed cell group (Fig. 5B,C, OC), and empty and TGF-β treatment group (Fig. 5B,C, ET).In Figs.1E and 2B, TGF-β treatment enhanced the expressions of Kv3.4,N-cadherin, and vimentin, while the expression level of E-cadherin was reduced.These results were also confirmed by the treatment of TGF-β in Kv3.4 overexpressed cells, especially in N-cadherin (Fig. 5D-H).To assess the synergistic effect of Kv3.4 and TGF-β in cell migration, we performed the transwell migration assay (Fig. 5I,J).Consistent with these results, Kv3.4 overexpressed cells with TGF-β treatment apparently enhanced the cell migration.Therefore, our results indicate that Kv3.4 could have a considerable synergistic effect with TGF-β.

Upregulated KCNC4 expression is associated with poor overall survival in human lung adenocarcinoma and squamous cell carcinoma patients
To investigate the role of Kv3.4 in human lung cancer, we performed a Kaplan-Meier (KM) analysis of Kv3.4 mRNA levels in lung cancer patients' overall survival in adenocarcinoma and squamous cell carcinoma (Fig. 6) 25,26 .Lung adenocarcinoma was the most common subtypes in worldwide in 2020, and squamous cell carcinoma accounted for 25% of cases in males and 12% in females, followed by adenocarcinoma (39% for males and 57% for females) 27 .According to the KM plotter of KCNC4, high expression levels of KCNC4 decreased overall survival in both adenocarcinoma (Fig. 6A, hazard ratio (HR) = 1.62, 95% confidence intervals (CI) = 1.25-2.11,log rank P = 0.00025, n = 1105) and squamous cell carcinoma (Fig. 6B, HR = 1.44, 95% CI = 1.04-1.98,log rank P = 0.025, n = 444).Therefore, we conclude that Kv3.4 expression is correlated with survival rates in human lung adenocarcinoma and squamous cell carcinoma.

Discussion
Kv3.4 is known to be related to cancer metastasis through cell migration and invasion 16 .EMT is a major mechanism for explaining cancer metastasis 4 .However, the relationship between Kv3.4 and EMT remains unclear.In this study, using TGF-β-induced EMT model in A549 cells, we confirmed the effects of cell migration and invasion by the regulation of Kv3.4.Our results showed that TGF-β treatment increased the expression levels of Kv3.4.The knockdown of Kv3.4 blocked the progression of EMT and apparently reduced cell migration and invasion.In contrast, the Kv3.4 overexpressed cells acquired mesenchymal characteristics retaining E-cadherin and promoted cell migration and invasion.Moreover, Kv3.4 overexpression had a synergistic effect with TGF-β treatment.In addition, analyzed by the KM plotter, high expression levels of Kv3.4 were considerably associated with poor overall survival of human lung adenocarcinoma and squamous cell carcinoma patients.Therefore, we conclude that Kv3.4 closely regulates cancer migration and invasion via TGF-β-induced EMT.EMT is a reversible biological program that converts epithelial cells to mesenchymal cells.In the process of EMT, immotile and apical-basal polarized cells get motile and front-back polarity, which could help cell migration, invasion, and cancer metastasis 1,6,7 .TGF-β is known to be one of the major inducers of EMT 24 .Our data show that TGF-β increased the expression levels of Kv3.4 and had a synergistic effect in Kv3.4 overexpressed cells.It has been known that TGF-β could modulates the expressions and functions of Kv channels.TGF-β increased the expression level of Kv2.1, which induced the maturation of rat primary cerebellar granule neurons 28 .Kv4.2 expression level was increased to transform from rat vascular fibroblasts to myofibroblasts 29 , but decreased in adult rat ventricular myocytes to protect myocytes from hypoxia or ischemia-reperfusion injuries 30 .
We found that the regulation of Kv3.4 induced partial EMT, which supports that Kv3.4 is closely related to cell migration and invasion.EMT is not a process of binary switches from fully epithelial to fully mesenchymal extremes.Instead, cancer cells transit intermediate epithelial/mesenchymal (E/M) states, or partial EMT, during metastatic cascades 6,8,31 .During partial EMT, representative epithelial and mesenchymal markers are exchanged and accompanied by morphological changes, migration and invasion, regulation of various signaling pathways and transcription factors.Therefore, these intermediate states provide advantages to cancer cells associated with phenotypic plasticity and heterogeneity 8 .Previous studies have shown that several Kv channels are involved in EMT, including Kv7.1 20 , Kv10.1 21 and Kv11.1 14,22,23 .The loss of Kv7.1 promoted EMT related with β-catenin 20 .In contrast, TGF-β-induced EMT enhanced the expression level of Kv10.1 in A549 cells 21 .In addition, the activation of Kv11.1 inhibited EMT and blocked the cancer metastasis by β-catenin in breast cancer 14 .It is also demonstrated that the knockdown of Kv11.1 reversed EMT in colorectal cancer 22 and Kv11.1 promoted EMT, resulting in the migration and invasion through PI3K/AKT pathway in esophageal squamous cell carcinoma 23 .In the present study, our data also suggest the possibility that Kv3.4 could be related to N-cadherin, a representative EMT marker that is closely involved in cancer metastasis, especially in cell migration 32 .According to previous studies, Kv channels are associated with N-cadherin.Kv1.5 and N-cadherin are colocalized in human cardiac myocytes, which enhances Kv1.5 activity 33 .In addition, the expression of Kv1.5 was apparently reduced in N-cadherin conditional knockout mouse ventricular myocytes 34 .Thus, further studies might be needed to reveal the precise mechanism between Kv channels and EMT, especially Kv3.4.
Recent studies have revealed the diverse roles of Kv3.4.First, Kv3.4 has been linked to the several signaling pathways.As an oxidation-and hypoxia-sensitive channel 35 , Kv3.4 is related to hypoxia-inducing factor 1α (HIF-1α), which binds to the Kv3.4 promoter 36 .Kv3.4 is also associated with calcium signaling, inducing calmodulin/calmodulin-dependent kinase-II axis 37 .Moreover, Kv3.4 is involved to AKT 15 and extracellular signal-regulated kinase (ERK) signaling pathway 16 as well.TGF-β is well-known as related to Mothers against decapentaplegic homolog (SMAD) signaling pathway 24,38 .However, our data revealed that the knockdown of Kv3.4 did not affect SMAD signaling (Supplement Fig. 2).Therefore, Kv3.4 might be related to SMAD-independent pathway in TGF-β-induced EMT 38,39 .Next, this study supports that Kv3.4 expression is closely involved with cancer malignancy.In addition to the cancer, Kv3.4 is also associated with various diseases such as cataracts 40

Cell circularity index for the quantification of the cell morphology
The morphology of cell was quantified by using the circularity index on the Fiji ImageJ software (National Institutes of Health, Maryland, USA).The circularity index formula is Circularity = 4 × π × (area)/(perimeter) 2 and the circularity index ranges from 0 to 1; A value = 1 indicates for a round shape, and 0 for an elongated morphology.

TGF-β treatment to induce the EMT
Using A549 cells, we treated TGF-β (Sigma-Aldrich, #T7039).Before the treatment of TGF-β, cells were starved with 0.1% FBS for 24 h 43 .After starvation, we treated 5 ng/ml TGF-β diluted with 2 mg/ml Bovine Serum Albumin (BSA, Sigma-Aldrich, #A9647) for 48 h with 0.1% FBS.In the case of the combination with TGF-β and Kv3.4 overexpression, Kv3.4-overexpressed cells were also starved with 0.1% FBS with G-418 for 24 h.And then, 2 ng/ml of TGF-β was treated in the cells for 24 h 44 .After the treatment, the cells were prepared for RNA or protein extraction.

Real-time PCR
Real-time PCR was performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, California, USA) using a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference.The real-time PCR reaction was Vol:.( 1234567890

Cell migration assay and invasion assay
The properties of cell migration and invasion were performed using a 24-well hanging insert (SPL, #35224) for transwell migration and invasion assay 46 .Before seeding the cells in a hanging well, cells were pretreated with siRNA or overexpression with Kv3.4 or TGF-β for the treatment group, and control siRNA or control vector or 2 mg/ml BSA for the control group.After cell detachment, the appropriate numbers of cells were plated on the upper side of a cell-permeable membrane with FBS-free media, and media containing 10% FBS were placed on the lower chamber of the membrane.Following incubation of 24 h for migration, the migrated cells were stained using Diff-Quik staining (Sysmex Corporation, Hyogo, Japan, #38721) and imaged by microscope (CKX53, Olympus).For invasion assay, 120 μl of 1.0 mg/ml diluted Matrigel (Corning ® , Arizona, USA, #356234) was coated on the upper insert membrane for 1 h at 37 °C before cell plating.After plating the appropriate numbers of cells for 48 h, invaded cells were stained using Diff-Quik staining, and imaged.Migrated and invaded cells

Kaplan-Meier analysis of overall survival in human lung adenocarcinoma and squamous cell carcinoma patients in silico
Using the online KM plotter lung cancer database (https:// kmplot.com/ analy sis, 2023.04.20), we generated overall survival KM plotters with KCNC4 (Affymetrix ID: 228436_at) split patients by 'auto select best cutoff ' in both lung adenocarcinoma (n = 1105) and squamous cell carcinoma (n = 444) analyzed by univariate cox regression 26 .

Statistics and reproducibility
Results were representative of at least three independent experiments, and all data were presented as the mean ± standard error.In case of passing the Kolmogorov-Smirnov (KS) normality test, we applied the unpaired t test for the statistical analysis of data; if not, we used the Mann-Whitney U test for statistical comparisons between two groups (GraphPad Prism Version 5.0, GraphPad Software, California, USA).Among the groups, we used one-way ANOVA with Tukey's multiple comparison test for post-hoc test for passing KS normality test; if not, we used the Kruskal-Wallis test with Dunn's multiple comparison test for post-hoc test (GraphPad Prism Version 6.0, GraphPad Software).

Figure 2 .
Figure 2. TGF-β treatment increased the expression levels of Kv3.4.(A) Representative graph shows the expression level of Kv3.4 in mRNA level after treatment with 5 ng/ml TGF-β for 48 h in A549 cells.(B,C) The protein level of Kv3.4 in A549 cells was analyzed by western blot (B) and the quantitative graph (C) after the TGF-β treatment.All experiments were repeated (n = 5), and the data represent the mean ± standard error.**p < 0.01; ***p < 0.001 (unpaired t test).

Figure 6 .
Figure 6.Upregulated KCNC4 expression is associated with poor overall survival in human lung adenocarcinoma and squamous cell carcinoma patients.KM analysis of the correlation between KCNC4 expression and overall survival for human lung adenocarcinoma (A, n = 1105) and squamous cell carcinoma patients (B, n = 444) using the online KM plotter lung cancer database (univariate Cox regression).

Table 1 .
Real-time PCR primer sets.

Table 2 .
Antibody information.www.nature.com/scientificreports/were randomly photographed in five fields per each membrane and quantified with Fiji ImageJ software (National Institutes of Health).